Resonator for detecting single molecule binding

ABSTRACT

Various embodiments of an apparatus for measuring binding kinetics of an interaction of an analyte material present in a fluid sample are disclosed. The apparatus includes a sensing resonator having at least one binding site for the analyte material; actuation circuitry adapted to drive the sensing resonator into an oscillating motion; measurement circuitry coupled to the sensing resonator and adapted to measure an output signal of the sensing resonator representing resonance characteristics of the oscillating motion of the sensing resonator; and a controller coupled to the actuation and measurement circuitry, wherein the controller is adapted to detect an individual binding event between the at least one binding site and a molecule of the analyte material.

RELATED APPLICATIONS

This application is the § 371 U.S. National Stage of InternationalApplication No. PCT/US2017/029311, filed on Apr. 25, 2017, which claimsthe benefit of U.S. Provisional Patent Application No. 62/327,749, filedon Apr. 26, 2016, the disclosures of which are hereby incorporatedherein by reference in their entireties to the extent that it does notconflict with the disclosure presented herein.

BACKGROUND

Piezoelectric devices such as thin film bulk acoustic wave (BAW)resonators and similar technologies like quartz crystal microbalances(QCM) have been employed as mass detectors for some time. Oneapplication of piezoelectric resonators is in detecting very smallquantities of materials. Piezoelectric resonators used as sensors insuch applications are sometimes called “micro-balances.” A piezoelectricresonator is typically constructed as a thin, planar layer ofcrystalline or polycrystalline piezoelectric material sandwiched betweentwo electrode layers. When used as a sensor, the resonator is exposed tothe material being detected to allow the material to bind on a surfaceof the resonator.

One conventional way of detecting the amount of the material bound onthe surface of a sensing resonator is to operate the resonator at itsresonant frequency in an oscillator circuit. As the material beingdetected binds on the resonator surface, the oscillation frequency ofthe resonator is reduced. The change in the oscillation frequency of theresonator, presumably caused by the binding of the material on theresonator surface, is measured and used to calculate the amount of thematerial bound on the resonator or the rate at which the materialaccumulates on the resonator surface.

The sensitivity of a piezoelectric resonator in air as a material sensoris theoretically proportional to the square of the resonance frequency.Thus, the sensitivities of material sensors based on the popular quartzcrystal resonators are limited by their relatively low oscillatingfrequencies, which typically range from several MHz to about 100 MHz.The development of thin-film resonator (TFR) technology can potentiallyproduce sensors with significantly improved sensitivities. A thin-filmresonator is formed by depositing a thin film of piezoelectric material,such as AlN or ZnO, on a substrate. Due to the small thickness of thepiezoelectric layer in a thin-film resonator, which is on the order ofseveral microns, the resonant frequency of the thin-film resonator is onthe order of 1 GHz. The high resonant frequencies and the correspondinghigh sensitivities make thin-film resonators useful for material sensingapplications. However, mass sensitivity of even thin-film resonators maybe limited for detection of certain analytes, such as biologicalanalytes.

The use of piezoelectric resonator sensors in immunoassays has beendescribed previously. In general, piezoelectric based immunoassays, inwhich mass change is attributable to the immunological reaction betweenan antigen and an antibody, can in some circumstances suffer from poorsensitivity and poor detection limit.

SUMMARY

In one aspect, the present disclosure provides an apparatus formeasuring binding kinetics of an interaction of an analyte materialpresent in a fluid sample. The apparatus includes a sensing resonatorhaving at least one binding site for the analyte material; actuationcircuitry adapted to drive the sensing resonator into an oscillatingmotion; measurement circuitry coupled to the sensing resonator andadapted to measure an output signal of the sensing resonatorrepresenting resonance characteristics of the oscillating motion of thesensing resonator; and a controller coupled to the actuation andmeasurement circuitry, where the controller is adapted to detect anindividual binding event between the at least one binding site and amolecule of the analyte material.

In another aspect, the present disclosure provides a method carried outby an apparatus for measuring binding kinetics of an interaction of ananalyte material present in a fluid sample. The method includescontacting a sensing resonator with the fluid sample, where the sensingresonator includes at least one binding site for the analyte material;actuating the sensing resonator into an oscillating motion; measuring anoutput signal representing resonance characteristics of the oscillatingmotion of the sensing resonator; and detecting an individual bindingevent between the at least one binding site and a molecule of theanalyte material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are schematic diagrams illustrating the operationalprinciples of embodiments of thin film bulk acoustic wave (BAW)resonator sensing devices.

FIG. 2 is a schematic diagram showing components of a BAW system fordetecting an analyte.

FIGS. 3A-B are schematic diagrams of an embodiment of a first bindingpartner bound to a surface of a BAW sensor (3A) and a recognitioncomponent bound to a second binding partner, which is bound to the firstbinding partner (3B).

FIG. 4 is a representative curve showing the frequency of a resonancepoint of a BAW sensor over time as molecules are binding.

FIG. 5 is a representative curve of a frequency shift spectrumassociated with multiple binding events of an analyte to a BAW sensor.

The schematic drawings are not necessarily to scale. Like numbers usedin the figures refer to like components, steps and the like. However, itwill be understood that the use of a number to refer to a component in agiven figure is not intended to limit the component in another figurelabeled with the same number. In addition, the use of different numbersto refer to components is not intended to indicate that the differentnumbered components cannot be the same or similar.

DETAILED DESCRIPTION

In the following detailed description several specific embodiments ofcompounds, compositions, products and methods are disclosed. It is to beunderstood that other embodiments are contemplated and may be madewithout departing from the scope or spirit of the present disclosure.The following detailed description, therefore, is not to be taken in alimiting sense.

This disclosure generally relates to, among other things, methods,devices, sensors, and systems for detecting an analyte. The methods,devices, sensors, and systems use a thin film bulk acoustic wave (BAW)resonator that measures a change in frequency or phase of the resonatorcaused by the binding of the analyte on a surface of the resonator. Aninput electrical signal having a phase and having a frequency within aresonance band of the piezoelectric resonator, which in the case of someembodiments of the present disclosure may be about 500 MHz or greater,such as about 1.5 GHz or greater, is coupled to and transmitted throughthe resonator to generate an output electrical signal which isfrequency-shifted or phase-shifted from the input signal due to binding,deposition, etc. of material being detected on the resonator surface.The output electrical signal received from the piezoelectric resonatoris analyzed to determine the change in frequency or phase caused by thebinding of analyte on the resonator surface. The measured change infrequency or phase provides quantitative information regarding theanalyte (or tag-linked analyte molecule) bound to the resonator surface.

Sensors, Devices and Systems

The sensors disclosed herein include at least one thin film resonatorsensor, such as a thin film bulk acoustic wave (BAW) resonator sensor. ABAW sensor includes a piezoelectric layer, or piezoelectric substrate,and input and output transducers. BAW sensors are small sensors, makingthe technology suitable for use in handheld devices. Accordingly, ahandheld device for detecting target analytes comprising a sensordescribed herein is contemplated.

Turning now to the drawings with reference to FIGS. 1A and 1B, generaloperating principles of an embodiment of a bulk-acoustic wavepiezoelectric resonator 20 used as a sensor to detect an analyte areshown. The resonator 20 typically includes a planar layer ofpiezoelectric material bounded on opposite sides by two respective metallayers that form the electrodes of the resonator. The two surfaces ofthe resonator are free to undergo vibrational movement when theresonator is driven by a signal within the resonance band of theresonator. When the resonator is used as a sensor, at least one of itssurfaces is adapted to provide binding sites for the material beingdetected. The binding of the material on the surface of the resonatoralters the resonant characteristics of the resonator, and the changes inthe resonant characteristics are detected and interpreted to providequantitative information regarding the material being detected.

By way of example, such quantitative information may be obtained bydetecting a change in the insertion or reflection coefficient phaseshift of the resonator caused by the binding of the material beingdetected on the surface of the resonator. Such sensors differ from thosethat operate the resonator as an oscillator and monitor changes in theoscillation frequency. Rather such sensors insert the resonator in thepath of a signal of a pre-selected frequency and monitor the variationof the insertion or reflection coefficient phase shift caused by thebinding of the material being detected on the resonator surface. Ofcourse, sensors that monitor changes in oscillation frequency may alsobe employed in accordance with the methods described herein.

In more detail, FIG. 1A shows the resonator 20 before the material beingdetected is bound to its surface 26. The depicted resonator 20 iselectrically coupled to a signal source 22, which provides an inputelectrical signal 21 having a frequency f within the resonance band ofthe resonator. The input electrical signal is coupled to the resonator20 and transmitted through the resonator to provide an output electricalsignal 23. In the depicted embodiment, the output electrical signal 23is at the same frequency as the input signal 21, but differs in phasefrom the input signal by a phase shift ΔΦ₁, which depends on thepiezoelectric properties and physical dimensions of the resonator. Theoutput signal 23 is coupled to a phase detector 24 that provides a phasesignal related to the insertion phase shift.

FIG. 1B shows the sensing resonator 20 with the material being detectedbound on its surface 26.

The same input signal is coupled to the resonator 20. Because theresonant characteristics of the resonator are altered by the binding ofthe material as a perturbation, the insertion phase shift of the outputsignal 25 is changed to ΔΦ₂. The change in insertion phase shift causedby the binding of the material is detected by the phase detector 24. Themeasured phase shift change is related to the amount of the materialbound on the surface of the resonator.

FIG. 1C shows an alternative to measuring the insertion phase of theresonator. A directional coupler 27 is added between the signal source22 and the resonator 20 with the opposite electrode grounded. A phasedetector 28 is configured to measure the phase shift of the reflectioncoefficient as a result of material binding to the resonator surface.

Other BAW phase-shift sensors that may be employed with the aspectsdescribed herein include those described in, for example, U.S. Pat. No.8,409,875, entitled RESONATOR OPERATING FREQUENCY OPTIMIZATION FORPHASE-SHIFT DETECTION SENSORS, which patent is hereby incorporatedherein by reference in its entirety to the extent that it does notconflict with the disclosure presented herein. For example, sensorapparatuses may include (i) a sensing resonator including binding sitesfor an analyte; (ii) actuation circuitry configured to drive the sensingresonator in an oscillating motion; (iii) measurement circuitry arrangedto be coupled to the sensing resonator and configured to measure one ormore resonator output signals representing resonance characteristics ofthe oscillating motion of the sensing resonator; and (iv) a controlleroperatively coupled with the actuation and measurement circuitry. Thecontroller can be interfaced with data storage containing instructionsthat, when executed, cause the controller to adjust the frequency atwhich the actuation circuitry drives the sensing resonator to maintain aresonance point of the sensing resonator. Accordingly, sensing may beaccomplished by actuating the BAW sensor into an oscillating motion;measuring one or more resonator output signals representing resonancecharacteristics of the oscillating motion of the BAW sensor; andadjusting the actuation frequency of the sensing resonator to maintain aresonance point of the BAW sensor.

Such phase detection approaches can be advantageously used withpiezoelectric resonators of different resonant frequencies.

In various embodiments, BAW sensors for use with the methods, devices,and system described herein have resonance frequencies of about 500 MHzor greater, such as about 700 MHz or greater, about 900 MHz or greater,about 1 MHz or greater, about 1.5 GHz or greater, about 1.8 GH orgreater, about 2 GHz or greater, about 2.2 GHz or greater, about 2.5 GHzor greater, about 3 GHZ or greater, or about 5 GHZ or greater canprovide enhanced sensitivity. In embodiments, the BAW sensors haveresonance frequencies of from about 500 MHz to about 5 GHz, such as fromabout 900 MHz to about 3 GHz, or from about 1.5 GHz to about 2.5 GHz.Some of such frequencies are substantially higher than frequencies ofpreviously described piezoelectric resonators.

The sensing resonators described herein are thin-film resonators. Thinfilm resonators include a thin layer of piezoelectric material depositedon a substrate, rather than using, for example, AT-cut quartz. Thepiezoelectric films typically have a thickness of less than about 5micrometers, such as less than about 2 micrometers, and may havethicknesses of less than about 100 nanometers. Thin-film resonators aregenerally preferred because of their high resonance frequencies and thetheoretically higher sensitivities. Depending on the applications, athin-film resonator used as the sensing element may be formed to supporteither longitudinal or shear bulk-acoustic wave resonant modes.Preferably, the sensing element is formed to support shear bulk-acousticwave resonant modes, as they are more suitable for use in a liquidsample.

Additional details regarding sensor devices and systems that may employTFRs are described in, for example, U.S. Pat. No. 5,932,953 issued Aug.3, 1999 to Drees et al., which patent is hereby incorporated herein byreference in its entirety to the extent that it does not conflict withthe disclosure presented herein.

TFR sensors may be made in any suitable manner and of any suitablematerial. By way of example, a resonator may include a substrate such asa silicon wafer or sapphire, a Bragg mirror layer or other suitableacoustic isolation means, a bottom electrode, a piezoelectric material,and a top electrode.

Any suitable piezoelectric material may be used in a TFR. Examples ofsuitable piezoelectric substrates include lithium tantalate (LiTaO₃),lithium niobate (LiNbO₃), Zinc Oxide (ZnO), aluminum nitride (AlN),plumbum zirconate titanate (PZT) and the like.

Electrodes may be formed of any suitable material, such as aluminum,tungsten, gold, titanium, molybdenum, or the like. Electrodes may bedeposited by vapor deposition or may be formed by any other suitableprocess.

Any suitable device or system may employ a thin film resonator asdescribed herein. By way of example and with reference to FIG. 2, asystem or apparatus for detecting an analyte may include a container 10(or more than one container), the thin film resonator 20, actuationcircuitry 22, measurement circuitry 29, and control electronics or acontroller 30. A fluid path couples the one or more containers 10 to theresonator 20. The control electronics 30 are operably coupled to theactuation circuitry and the measurement circuitry. In embodiments,control electronics 30 are configured to modify the frequency at whichthe actuation circuitry 22 oscillates the resonator 20 based on inputfrom the measurement circuitry 29.

Any suitable control electronics or controller 30 may be employed. Forexample, control electronics may include a processor, controller,memory, or the like. Memory may include computer-readable instructionsthat, when executed by processor or controller cause the device andcontrol electronics to perform various functions attributed to deviceand control electronics described herein. Memory may include anyvolatile, non-volatile, magnetic, optical, or electrical media, such asa random access memory (RAM), read-only memory (ROM), non-volatile RAM(NVRAM), electrically-erasable programmable ROM (EEPROM), flash memory,or any other digital media. Control electronics 30 may include any oneor more of a microprocessor, a controller, a digital signal processor(DSP), an application specific integrated circuit (ASIC), afield-programmable gate array (FPGA), or equivalent discrete orintegrated logic circuitry. In some examples, control electronics 30 mayinclude multiple components, such as any combination of one or moremicroprocessors, one or more controllers, one or more DSPs, one or moreASICs, or one or more FPGAs, as well as other discrete or integratedlogic circuitry. The functions attributed to control electronics hereinmay be embodied as software, firmware, hardware or any combinationthereof.

Referring now to FIGS. 3A-B, a first molecular recognition component 100may be bound to a surface 26 of a BAW sensor 20 via one or moreintermediates. For example, a first binding partner 99 may be bound tothe surface 26 and the first molecular recognition component 100 mayinclude a second binding partner 101 configured to selectively bind tothe first binding partner 99. First recognition component 100 may bebound to surface 26 via binding partners 99, 101 at any suitable time,such as before the sensor 20 is incorporated into a device or system orafter the sensor 20 is incorporated into the device or system. Forexample, first recognition component 100 may be bound to surface 26 viabinding partners 99, 101 as a first step of, or during, an analytedetection assay.

One or more embodiments of systems and apparatuses described herein canbe utilized to detect an individual binding event between a molecule ofan analyte material and a binding site of a sensing resonator of theapparatus. In one or more embodiments, such apparatus can be utilized todetermine a binding rate of the analyte material, e.g., by countingindividual binding events. By way of example, FIG. 4 shows arepresentative curve of the frequency of a resonance point of a sensorover time in such apparatus. One or more embodiments of an apparatus candiscriminate between binding and non-binding sources, e.g., bydetermining a frequency shift of the sensing resonator caused by theindividual binding event. For example, section 202 of the curve in FIG.4 shows the frequency shift caused by the individual binding event.Sections 204, 206, and 208 also show frequency shifts caused byindividual binding events. Further, for example, in one or moreembodiments, slow drift sources, as indicated by section 201 of thecurve in FIG. 4, can be filtered out by analyzing such frequency shifts.Further, in one or more embodiments, an apparatus can discriminatebetween different binding events (i.e. specific and non-specific)through a frequency shift spectrum of all individual binding events.FIG. 5 shows a representative curve of such a frequency shift spectrumof individual binding events of an analyte to a resonant sensor. Thepeak and spread of such a spectrum is unique to analytes of differentmass and size.

The following equations can be utilized to detect individual bindingevents:

Simplified Binding Equation

d/dt[Bound]_(surface) B ₀−Bound]_(surface)[Sample]k _(a)if [Bound]_(surface) is <<[B ₀]_(surface):d/dt[Bound]_(surface) B ₀]_(surface)[Sample]k _(a),

Where B₀ is an unbound surface antibody at the start of binding (i.e.,the surface is 100% unbound at the beginning of the process);

“Bound” is a surface antibody bound to an antigen;

“Sample” is the sample containing the antigen to be measured;

k_(a) is the on-rate of the antibody-antigen pair;

[ ] represents a volume concentration; and

[ ]_(surface) represents a surface concentration.

Sauerbrey Equation

${\Delta\; f} = {{- \frac{2f_{0}^{2}}{A\sqrt{\rho_{q}\mu_{q}}}}\Delta\;{m.}}$

From these equations, the following can be observed:

(1) Frequency shift is dependent on binding density(Δm/A=[Bound]_(surface)) and is independent of resonator active area (A)for a given coating density;

(2) The inverse of resonator area (1/A) acts like the gain. As areadecreases, frequency shift/mass goes up;

(3) If resonator area is sufficiently reduced, an individual bindingevent can be detected as a discrete frequency shift above the systemnoise/drift. This would be a single-molecule detector. Individualbinding events can be counted over time, and the rate of such bindingevents can be related back to the concentration of the sample;

(4) Binding events can produce a rapid frequency shift that can beeasily distinguished from slower components produced, e.g., bytemperature, fluid shear, or others;

(5) Since the frequency shift associated with each individual bindingevent is still proportional to mass, if total noise is low enough, abinding spectrum (counts vs. frequency shift) can be constructed thatcan further distinguish between different types of binding events (i.e.non-specific binding vs. specific binding). For example, a non-specificserum protein binding may bind with a larger average shift than thetarget binding event. If there is enough statistical separation, thenon-specific binding could be separated out from the specific binding.This technique can also be used to correct double-counts that mightoccur during dead-time;

(6) At small resonator areas and low concentrations, binding events perminute will be extremely low and one resonator alone may not record anyor enough events within a reasonable capture time. Arrays can beconstructed from individual resonators to create a large enoughaggregate detector area to count sufficient binding events during theallotted capture time. Each individual resonator in the array can beindependently driven and measured. In this case, detection limit scaleswith array size. The larger the array, the lower the detection limit,provided the readout electronics are capable of interrogating the arrayfast enough. (This assumes that non-specific binding can be effectivelymanaged);

(7) Adaptive readout schemes may be utilized that read a small number ofresonators extremely quickly when event rates are high (e.g., for highconcentration samples), and a large number of resonators more slowlywhen event rates are low (e.g., low concentration samples).

The size of a capture antibody can be estimated at 15 nm×15 nm×3 nm(Thermo Fisher Application note D19559). It can be immobilized on a flatsurface 100% oriented (footprint=15 nm×3 nm) or 100% non-oriented(footprint=15 nm×15 nm), or more typically something in between.

Binding saturation on aluminum nitride 2 GHz BAW shear mode devices istypically on the order of 750 kHz for direct binding (this is antigendependent). Device improvements to the BAW suggest that the masssensitivity can be improved up to 10 fold. In this case, it would beexpected to saturate near 7.5 MHz.

The level of orientation of antibody immobilization on the BAW surfaceand the packing density of the bound antigen may be unknown. However,the antibody coating should fall between the 100% oriented surface and100% non-oriented surface. These conditions can be used as an upper andlower bound to estimate the active area of the BAW resonator needed todetect single binding events.

Non-oriented, Oriented, High Low Density Density (15 nm x 15 nm (15 nm x3 nm footprint) footprint) Current Device Saturation Shift (Hz) 750000750000 500 Hz Total Binding Sites 1500 1500 Binding Active Area (μm²)0.3375 0.0675 Event 100 Hz Total Binding Sites 7500 7500 Binding ActiveArea (μm²) 1.6875 0.3375 Event Improved Device Saturation Shift (Hz)7500000 7500000 500 Hz Total Binding Sites 15000 15000 Binding ActiveArea (μm²) 3.375 0.675 Event 100 Hz Total Binding Sites 75000 75000Binding Active Area (μm²) 16.875 3.375 Event

The approximate resonator area needed to operate in binding eventcounting mode based on the total shift desired per event to overcomesystem noise can be estimated. Two different frequency shift thresholdscan be used: 500 Hz and 100 Hz. The current BAW device can be capable ofcounting binding events if the active area is approximately 1 μm². Theimproved BAW device can be capable of counting binding events if theactive area is approximately 4-17 μm².

Since at these extremely small device areas binding events will be rareat low analyte concentrations, an array may be needed to increase theeffective detection area. Each BAW resonator in the array is capable ofcounting binding events independently, and the events from each BAWresonator are summed to generate a total for the array. The count ratefor a 256 element array is estimated as a function of analyteconcentration. Using the current BAW response of 10 ng/ml rTSH in abuffer/BSA solution of approximately 5 kHz/min responses (in bindingevents/min) can be estimated for BAW devices with an area of 1.7 μm².

Current Sensor Events/ Events in Response min 10 min. 256 Array Sample(Fractional (Single (Single Total Concentration Response Saturation/ BAWA = BAW A = Events in (ng/ml) (Hz/min) min) 1.7 μm²) 1.7 μm²) 10 min. 105000 0.0066667 50.00 500 128000 1 500 0.0006667 5.00 50 12800 0.1 500.0000667 0.50 5 1280 0.01 5 0.0000067 0.05 0.5 128 0.001 0.5 0.00000070.01 0.05 12.8 Improved Sensor (approx. 10x mass sensitivity) Events/Events in Response min 10 min. 256 Array Sample (Fractional (Single(Single Total Concentration Response Saturation/ BAW A = BAW A = Eventsin (ng/ml) (Hz/min) min) 17 μm²) 17 μm²) 10 min. 10 50000 0.0066667500.00 5000 1280000 1 5000 0.0006667 50.00 500 128000 0.1 500 0.00006675.00 50 12800 0.01 50 0.0000067 0.50 5 1280 0.001 5 0.0000007 0.05 0.5128

In one aspect, the present disclosure provides an apparatus formeasuring binding kinetics of an interaction of an analyte materialpresent in a fluid sample. The apparatus includes a sensing resonatorhaving at least one binding site for the analyte material. Any suitablesensing resonator can be utilized. Further, any suitable number ofsensing resonators can be utilized. In one or more embodiments, thesensing resonator can include an array of sensing resonators. In one ormore embodiments, the sensing resonator is a bulk acoustic waveresonator that can include a resonant frequency of at least 900 MHz andno greater than 10 GHz. In one or more embodiments, the sensingresonator is of sufficiently small mass for the controller to detect theindividual binding event. As used herein, the term “sufficiently small”means that the device area is selected to be small enough to provide ameasureable frequency shift above the noise level due to a singlebinding event. In one or more embodiments, the mass of the sensingresonator can be reduced to increase the sensitivity of the apparatus.

The apparatus also includes actuation circuitry adapted to drive thesensing resonator into an oscillating motion. Any suitable actuationcircuitry can be utilized, e.g., the actuation circuitry describedherein. In one or more embodiments, the actuation circuitry can beadapted to individually drive each resonator of an array of resonatorsat the same or different frequencies of one or more additionalresonators.

The apparatus can also include measurement circuitry coupled to thesensing resonator and adapted to measure an output signal of the sensingresonator representing resonance characteristics of the oscillatingmotion of the sensing resonator. Any suitable measurement circuitry canbe utilized, e.g., the measurement circuitry described herein. In one ormore embodiments, the measurement circuitry can be adapted toindividually measure an output signal of each sensing resonator of anarray of sensing resonators. In one or more embodiments, the apparatuscan include an array of sensing resonators each including at least onebinding site for the analyte material, where the measurement circuitryis adapted to measure an output signal of each sensing resonator of thearray of sensing resonators that represents resonance characteristics ofthe oscillating motion of the respective sensing resonator.

The apparatus can also include a controller coupled to the actuation andmeasurement circuitry, where the controller is adapted to detect anindividual binding event between the at least one binding site and amolecule of the analyte material. The controller can be further adaptedto perform one or more of the following:

-   -   determine the frequency at which the actuation circuitry drives        the sensing resonator;    -   detect the individual binding event by determining a first        frequency of the output signal of the sensing resonator prior to        the binding event and a second frequency of the output signal        following the individual binding event;    -   detect the individual binding event by determining a frequency        shift of the output signal of the sensing resonator based upon        the first frequency and the second frequency;    -   detect the individual binding event by comparing the frequency        shift to a frequency shift threshold. The threshold can be any        suitable value. In one or more embodiments, the frequency shift        can be compared to a lower threshold and an upper threshold,        i.e., individual binding events can be associated with a        frequency shift that is at greater than or equal to a lower        limit and less than or equal to an upper limit. Such comparison        to one or both of a lower and upper limit can aid in filtering        out non-binding events;    -   determine a binding rate of the analyte material;    -   determine the binding rate of the analyte material by counting a        number of individual binding events per unit time; and    -   determine a concentration of the analyte material based upon the        binding rate of the analyte material.

The apparatus can also include one or more reference resonators coupledto the actuation circuitry and the measurement circuitry. Any suitablereference resonator can be included, e.g., the reference resonatorsdescribed herein. In one or more embodiments, the reference resonator isfree of binding sites of the analyte material. In one or moreembodiments, the actuation circuitry is adapted to independently drivethe reference resonator and the sensing resonator into oscillatingmotion.

Any suitable technique or combination of techniques can be utilized withthe systems and apparatuses described herein to measure the bindingkinetics of an interaction of an analyte material present in a fluidsample and, e.g., to detect an individual binding event. In one or moreembodiments, a sensing resonator can be contacted with the fluid sample,where the sensing resonator includes at least one binding site for theanalyte material. The sensing resonator can be actuated into anoscillating motion. An output signal representing resonancecharacteristics of the oscillating motion of the sensing resonator canbe measured. Further, an individual binding event between the at leastone binding site and a molecule of the analyte material can be detected.In one or more embodiments, detecting the individual binding event caninclude detecting a frequency shift of the output signal of the sensingresonator.

In one or more embodiments, the frequency shift of the output signal canbe detected by determining a first frequency of the output signal of thesensing resonator prior to the binding event; determining a secondfrequency of the output signal of the sensing resonator following theindividual binding event; and comparing the first frequency to thesecond frequency. In one or more embodiments, the frequency shiftrepresentative of one or more subsequent binding events can be detectedusing any suitable technique or combination of techniques.

In one or more embodiments, the method can also include contacting areference resonator with the fluid sample, wherein the referenceresonator is free of binding sites for the analyte material; actuatingthe reference resonator into an oscillating motion; and measuring anoutput signal representing resonance characteristics of the oscillatingmotion of the reference resonator.

In one or more embodiments, the method can also include comparing theoutput signal of the sensing resonator to the output signal of thereference resonator.

In one or more embodiments, the method can include determining a bindingrate of the analyte material. In one or more embodiments, the bindingrate can be determined by counting a number of individual binding eventsper unit time. For example, in one or more embodiments, a number ofindividual binding invents that cause a first frequency shift can becounted, and a number of individual binding invents that cause a secondfrequency shift can also be counted. Additional counts of third, fourth,fifth, etc., frequency shifts can be counted, and a histogram orspectrum of counts of individual binding invents versus frequency shiftsor bands can be formulated. In one or more embodiments, a particularanalyte can exhibit a unique or characteristic histogram such that theparticular analyte can be determined based upon the histogram providedby the apparatus. In one or more embodiments, a particular analyte canexhibit a unique or characteristic histogram such that only bindingevents corresponding to that particular analyte are used to determine abinding rate.

Use

The sensors, devices, and systems described herein may be employed todetect an analyte in a sample. The sensors may find use in numerouschemical, environmental, food safety, or medical applications. By way ofexample, a sample to be tested may be acquired or may be derived fromblood, serum, plasma, cerebrospinal fluid, saliva, urine, and the like.Other test compositions that are not fluid compositions may be dissolvedor suspended in an appropriate solution or solvent for analysis.

Definitions

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” encompass embodiments having pluralreferents, unless the content clearly dictates otherwise.

As used in this specification and the appended claims, the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise. The term “and/or” means one or all of thelisted elements or a combination of any two or more of the listedelements.

As used herein, “have”, “having”, “include”, “including”, “comprise”,“comprising” or the like are used in their open ended sense, andgenerally mean “including, but not limited to”. It will be understoodthat “consisting essentially of”, “consisting of”, and the like aresubsumed in “comprising” and the like. As used herein, “consistingessentially of,” as it relates to a composition, product, method or thelike, means that the components of the composition, product, method orthe like are limited to the enumerated components and any othercomponents that do not materially affect the basic and novelcharacteristic(s) of the composition, product, method or the like.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the disclosure, including the claims.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc. or 10 or less includes 10, 9.4, 7.6, 5, 4.3,2.9, 1.62, 0.3, etc.). Where a range of values is “up to” a particularvalue, that value is included within the range.

Any direction referred to herein, such as “top,” “bottom,” “left,”“right,” “upper,” “lower,” and other directions and orientations aredescribed herein for clarity in reference to the figures and are not tobe limiting of an actual device or system or use of the device orsystem. Devices or systems as described herein may be used in a numberof directions and orientations.

“Binding event,” as used herein, means the binding of a target analyteto a molecular recognition component immobilized in a surface of asensor.

Thus, embodiments of A RESONATOR FOR DETECTING SINGLE MOLECULE BINDINGare disclosed. One skilled in the art will appreciate that the devicessuch as signal generators, systems, and methods described herein can bepracticed with embodiments other than those disclosed. The disclosedembodiments are presented for purposes of illustration and notlimitation. One will also understand that components of the leadsdepicted and described with regard the figures and embodiments hereinmay be interchangeable.

What is claimed is:
 1. An apparatus for measuring binding kinetics of aninteraction of an analyte material present in a fluid sample,comprising: a sensing resonator comprising at least one binding site forthe analyte material; actuation circuitry adapted to drive the sensingresonator into an oscillating motion; measurement circuitry coupled tothe sensing resonator and adapted to measure an output signal of thesensing resonator representing resonance characteristics of theoscillating motion of the sensing resonator; and a controller coupled tothe actuation and measurement circuitry, wherein the controller isadapted to detect an individual binding event between the at least onebinding site and a molecule of the analyte material.
 2. The apparatus ofclaim 1, wherein the controller is further adapted to determine thefrequency at which the actuation circuitry drives the sensing resonator.3. The apparatus of claim 1, wherein the controller is further adaptedto detect the individual binding event by determining a first frequencyof the output signal of the sensing resonator prior to the binding eventand a second frequency of the output signal following the individualbinding event.
 4. The apparatus of claim 3, wherein the controller isfurther adapted to detect the individual binding event by determining afrequency shift of the output signal of the sensing resonator based uponthe first frequency and the second frequency.
 5. The apparatus of claim4, wherein the controller is further adapted to detect the individualbinding event by comparing the frequency shift to a frequency shiftthreshold.
 6. The apparatus of claim 1, further comprising a referenceresonator free of binding sites for the analyte material, wherein thereference resonator is coupled to the actuation circuitry and themeasurement circuitry.
 7. The apparatus of claim 6, wherein theactuation circuitry is adapted to independently drive the referenceresonator and the sensing resonator into oscillating motion.
 8. Theapparatus of claim 1, wherein the sensing resonator is a bulk acousticwave resonator comprising a resonant frequency of at least 900 MHz andno greater than 10 GHz.
 9. The apparatus of claim 1, wherein the sensingresonator is of sufficiently small mass for the controller to detect theindividual binding event.
 10. The apparatus of claim 1, wherein thecontroller is further adapted to determine a binding rate of the analytematerial.
 11. The apparatus of claim 10, wherein the controller isadapted to determine the binding rate of the analyte material bycounting a number of individual binding events per unit time.
 12. Theapparatus of claim 10, wherein the controller is further adapted todetermine a concentration of the analyte material based upon the bindingrate of the analyte material.
 13. The apparatus of claim 1, wherein thesensing resonator comprises an array of sensing resonators eachcomprising at least one binding site for the analyte material, whereinthe measurement circuitry is adapted to measure an output signal of eachsensing resonator of the array of sensing resonators that representsresonance characteristics of the oscillating motion of the respectivesensing resonator.